Microrna patterns for the diagnosis, prognosis and treatment of melanoma

ABSTRACT

The present invention relates to methods for diagnosing, staging, prognosticating and treating melanoma based on evaluating the expression of specific patterns of oncogenic or suppressive microRNA (miR) molecules in a patient in need thereof.

FIELD OF THE INVENTION

The present invention relates to methods for diagnosing, staging,prognosticating and treating melanoma based on evaluating the expressionof specific patterns of oncogenic or suppressive microRNA (miR)molecules in a patient in need thereof

BACKGROUND OF THE INVENTION

Melanoma accounts for 4% of all skin cancer diagnoses but contributes to75% of deaths from skin cancer, where the incidence of melanoma anddeath rates from melanoma continue to rise, each year in the Westernworld.

In its early stages malignant melanoma can be cured by surgicalresection, but once it has progressed to the metastatic stage it isextremely difficult to treat. Diagnosis of melanoma is commonly based onpathology and immunological staining and prognostication is mainly basedon Breslow score (depth of invasion of the primary lesion).

MicroRNA (miR) molecules are major regulators of a large proportion ofanimal genes or the transcriptome. Association of various miRs withdiseases or susceptibility to disease is known in the art. For example,U.S. Patent Application, Publication No. 2007/0072204, discloses amethod for diagnosing a subject with various cancers based on theexpression level of mir17 (mir17-92 cistron).

Typical treatments of melanoma include surgery removal of the tumor,immunotherapy, chemotherapy and radiotherapy among others. The majorityof all melanomas are caused by a missense mutation (V600E) in the B-Rafoncogene. Thus, various agents targeted to the inhibition of the V600Egene product have been developed and include, for example, the compoundPLX4720 (known also as Vemurafenib), currently under human clinicalstudies. Additional novel approaches for melanoma therapy include agentswhich augment the anti-tumor immune response such as Ipilimumab, whichblocks CTLA4 inhibition of lymphocyts, thereby enhancing the immuneresponse directed to the tumor.

Creighton et al. (Cancer Res., Mar. 1, 2010; 70(5):1906-15) found miR-31to be under-expressed in ovarian cancer cell lines, and showed that itsforced expression induced p53-mediated apoptosis in different celllines.

It has been shown by Liu et al. (Clin. Cancer Res., 15:1177-1183, 2009)and others that miR-133 is down-regulated in various cancer cell lines.Others have shown that miR133a is a tumor suppressor in bladder cancer(Chiyomaru et al., British Journal of Cancer 102(5):883-91, 2010) and inesophageal cell carcinoma (Kano et al., Int. J. Cancer, Mar. 2, 2010,Epub: http://www.ncbi.nlm.nih.gov/pubmed/20198616).

MiR-184 was also found to be associated with squamous cell carcinoma oftongue (Wong et al., Clin. Cancer Res., 14(9):2588-92, 2008).

Lee et al. (PLoS Comput. Biol., Apr. 1, 2010, 6(4):e1000730) indicatesthat miR-204 is an onco-suppressor in head and neck tumor metastasis.

U.S. Patent Application Publication No. 2006/0105360 discloses a methodof diagnosing whether a subject has or is at risk of developing cancer,including melanoma, based on measuring the copy number of at least onemiR gene, including, miR-301.

U.S. Patent Application, Publication No. 2008/0026951, discloses theassociation of various miRs with different diseases, including theassociation of miR-17 with colon cancer; miR-31 with colon and thyroidcancers; miR-133a with cardiac hypertrophy and lupus; miR-184 with lupusand miR-204 with Alzheimer's disease.

U.S. Patent Application, Publication No. 2008/0306006, discloses theassociation of various miRs, including, miR-31; miR-34a; miR-184prec;miR-185 and miR-204, with solid tumors, specifically, breast cancer;lung cancer, prostate cancer, stomach cancer, colon cancer andpancreatic cancer.

According to U.S. Patent Application, Publication No. 2008/0076674 mi34aand miR185 are associated with breast cancer. Takahashi et al. (PLoSOne,4(8):e6677, 2009) discloses that miR185 induces cell cycle arrest inlung cancer cell lines.

U.S. Patent Application Publication No. 2009/0263803 discloses thatmiR-29a and miR-29c, are differentially expressed in lymph nodesobtained from melanoma patients compared to healthy control subjects.

A method for diagnosing a cancer, including melanoma, in a subject,comprising determining an amount of one or more miRs includingmiR-324-3p, is disclosed in U.S. Patent Application Publication No.2010/0196426, wherein if there is a measurable difference of said miR,the subject is diagnosed as having the cancer.

U.S. Pat. No. 7,897,356 discloses that miR-374, among other miRs, is aspecific biomarker for melanoma. However, according to US 2011/0107440,the expression of miR-374, miR-29a, miR-29c, miR-324-3p and miR-451,among other miRs, is indicative of non-melanoma skin cancer.

Identification of miRNAs that regulate the aggressive phenotype ofmelanoma cells is disclosed by the inventors of the present invention ina paper entitled: “Regulation of cancer aggressive features in melanomacells by microRNAs”, published after the priority date of the presentapplication (Greenberg et al. PLoS One. 2011 Apr. 25; 6(4):e18936).

There is an unmet need for effective method of diagnosing, staging,prognosticating and treating metastatic melanoma.

SUMMARY OF THE INVENTION

The present invention relates to methods for the diagnosis, staging andprognostication of melanoma and metastatic melanoma, as well as, fortreating metastatic melanoma, by evaluating the presence of patterns ofoncogenic and suppressive miRNA patterns that specifically relate to theonset or prevention of metastatic melanoma.

The present invention further discloses methods for diagnosis andstaging of melanoma by determining the presence of specific miRsexpression patterns. It is now disclosed for the first time thatspecific miR expression patterns indicative whether or not a subject hasor is susceptible to melanoma, are present in peripheral blood.

In addition, the present invention is based in part on the unexpecteddiscovery that particular miR patterns, which includes miRs known to begenerally associated with various diseases other than melanoma, wherefound to have specific roles in the suppression or oncogenesis ofaggressive types of melanoma. As exemplified below, the suppressive miRsof the invention inhibited the proliferation, invasion ability and theability to form tubes, in aggressive melanoma cells and tumors. It isfurther exemplified that the oncogenic miRs of the invention inducedsignificant proliferation of poorly aggressive melanoma cells, withrespect to control cells.

The present invention further discloses methods for predicting theresponsiveness to melanoma therapy. In particular, as disclosed herein,miRs identified in the peripheral blood of a subject having melanoma ormetastatic melanoma, may be used as predictors/biomarkers for theresponse of a subject to treatment with anti-cancer drugs, such as,Ipilimumab and Vemurafenib. Advantageously, the prediction of patient'sresponsiveness to melanoma therapy provides personalized medical tool,improves therapy management per patient, reduces exposure to sideeffects induced by unsuitable therapy and is cost effective among otheradvantages.

According to one aspect, the present invention provides a method fordiagnosing melanoma in a subject, comprising:

-   -   (a) obtaining from the subject a sample of peripheral blood        comprising RNA; and    -   (b) determining, in said RNA, the presence of a plurality of        oncogenic miRNAs, comprising miRNAs selected from the group        consisting of: miR-374a, miR-301a, SNORD-38b, miR-29c,        miR-324-3p, miR-451 and miR-29a,    -   wherein the presence of the plurality of oncogenic miRs is        indicative of the subject having, or being susceptible to        developing melanoma.

According to one embodiment, the plurality of oncogenic miRs furthercomprises one or more miRs selected from the group consisting of:miR-150, miR-342-3p, miR-197, miR-140-3p, miR-503, miR-339-3p,miR-628-3p and miR-20a*. Each possibility represents a separateembodiment of the invention.

According to another embodiment, the plurality of oncogenic miRs isselected from the group consisting of: miR-150, miR-451, miR-342-3p,SNORD38b, miR-324-3p, miR-29c, miR-197, miR-140-3p, miR-29a, miR-503,miR-339-3p, miR-628-3p, miR-20a*, miR-374a and miR-301a.

According to yet another embodiment, the plurality of oncogenic miRs isselected from the group consisting of: miR-374a, SNORD-38b and miR-301.Each possibility represents a separate embodiment of the invention.

According to yet another embodiment, the plurality of oncogenic miRscomprises SNORD-38b and one or more of miR-301 and miR-374. Eachpossibility represents a separate embodiment of the invention.

According to yet another embodiment, the plurality of oncogenic miRs isselected from the group consisting of: miR-374a, miR-29c, miR-324-3p,miR-451 and miR-29a.

According to yet another embodiment, the method comprises determiningthe presence of at least three oncogenic miRNA, or at least fouroncogenic miRNAs, the presence of which is indicative of the subjecthaving, or being susceptible to developing, melanoma. Each possibilityrepresents a separate embodiment of the invention.

According to yet another embodiment, the method comprises determiningthe presence of miR-374a, and at least one miR selected from the groupconsisting of miR-301a, SNORD-38b, miR-29c, miR-324-3p, miR-451,miR-29a, miR-150, miR-342-3p, miR-197, miR-140-3p, miR-503, miR-339-3pand miR-20a. Each possibility represents a separate embodiment of theinvention.

According to yet another embodiment, the plurality of oncogenic miRs areover-expressed at least 5 fold, at least 10 fold or at least 20 foldcompared to expression of said plurality of miRs a healthy subject. Eachpossibility represents a separate embodiment of the invention.

According to yet another aspect the present invention provides a methodfor diagnosing metastatic melanoma, comprising:

-   -   (a) obtaining a sample comprising RNA from a tumor of a subject;        and    -   (b) determining, in said RNA, the presence of a plurality of        oncogenic miRs comprising miR-17 and at least one additional        oncogenic miRNA selected from the group set forth in Table 1,    -   wherein the presence of the plurality of said oncogenic miRs is        indicative of the subject having, or being susceptible to        developing, metastatic melanoma.

TABLE 1 Oncogenic human (hsa) miRNAs Oncogenic miRs hsa-miR-603hsa-miR-15a* hsa-miR-302b* hsa-let-7f-2* hsa-miR-299-5p hsa-miR-19a*hsa-miR-7 hsa-miR-18a* hsa-miR-886-5p hsa-miR-183* hsa-miR-26b*hsa-miR-429 hsa-miR-886-3p hsa-miR-517b hsa-miR-142-5p hsa-miR-942hsa-miR-495 hsa-miR-29b-1* hsa-miR-190 hsa-miR-572 hsa-miR-137hsa-miR-30c-2* hsa-miR-17* hsa-miR-223 hsa-miR-600 hsa-miR-517ahsa-miR-193a-3p hsa-miR-892b hsa-miR-558 hsa-miR-373 hsa-miR-801hsa-miR-92a-1* hsa-miR-492 hsa-miR-941 hsa-miR-589 hsa-miR-616hsa-miR-127-5p hsa-miR-943 hsa-miR-29a* hsa-miR-182 hsa-miR-125b-1*hsa-miR-199b-5p hsa-miR-200b hsa-miR-99a* hsa-miR-21* hsa-miR-629hsa-miR-539 hsa-miR-604 hsa-miR-939 hsa-miR-106a hsa-miR-92ahsa-miR-125b hsa-miR-let-7e hsa-miR-222 hsa-miR-100 hsa-miR-15b*hsa-miR-661 hsa-miR-138 hsa-miR-18a hsa-miR-139-5p hsa-miR-210hsa-miR-30a* hsa-miR-99a hsa-miR-*222 hsa-miR-15b hsa-miR-let-7bhsa-miR-708 hsa-miR-935 hsa-miR-19b hsa-miR-18b hsa-miR-27a* hsa-miR-221hsa-miR-30e* hsa-miR-27a hsa-miR-*625 hsa-miR-142-3p hsa-miR-let-7ahsa-miR-29a hsa-miR-20a hsa-miR-*424 *miR generated from thecomplementary strand.

According to one embodiment, the method comprises determining thepresence of at least three oncogenic miRNA, or at least four oncogenicmiRNAs, wherein at least one oncogenic miRNA is miR-17, such that thepresence of which is indicative of the subject having, or beingsusceptible to developing, metastatic melanoma. Each possibilityrepresents a separate embodiment of the invention.

According to yet another embodiment, the second sample obtained from atumor comprises melanocytes.

According to yet another embodiment, the melanoma is selected from thegroup consisting of: lentigo maligna, lentigo maligna melanoma,superficial spreading melanoma, acral lentiginous melanoma, mucosalmelanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma,amelanotic melanoma and soft-tissue melanoma.

According to yet another aspect, the present invention provides a methodfor diagnosing melanoma in a subject, comprising

-   -   (a) obtaining from the subject a first sample of peripheral        blood comprising RNA;    -   (b) determining, in said RNA, the presence of a plurality of        miRNAs comprising miRNAs selected from the group consisting of        miR-374a, miR-301a, SNORD-38b, miR-29c, miR-324-3p, miR-451 and        miR-29a;    -   (c) identifying the subject as having, or being susceptible to,        melanoma; and    -   (d) obtaining from a tumor of said subject a second sample        comprising RNA and determining, in said RNA the presence of a        plurality of oncogenic miRs comprising miR-17 and at least one        additional miR selected from the group set forth in Table 1.        wherein the presence of the plurality of said oncogenic miRs is        indicative of the subject having, or being susceptible to        developing, metastatic melanoma.

According to one embodiment, the plurality of miRs further comprisesmiRs selected from the group consisting of: miR-150, miR-342-3p,miR-197, miR-140-3p, miR-503, miR-339-3p miR-628-3p and miR-20a*. Eachpossibility represents a separate embodiment of the invention.

According to another embodiment, the plurality of miRs is selected fromthe group consisting of: miR-374, SNORD-38b and miR-301. Eachpossibility represents a separate embodiment of the invention.

According to yet another embodiment, the plurality of miRs comprisesSNORD-38b and one or more of miR-301 and miR-374. Each possibilityrepresents a separate embodiment of the invention.

According to yet another embodiment, the plurality of miRs comprisesmiR-374, SNORD-38b and miR-301.

According to yet another embodiment, the plurality of miRs is selectedfrom the group consisting of: miR-374a, miR-29c, miR-324-3p, miR-451 andmiR-29a.

According to yet another embodiment, the plurality of miRs comprisesmiR-374a and at least one miR selected from the group consisting of:miR-301a, SNORD-38b, miR-29c, miR-324-3p, miR-451, miR-29a, miR-150,miR-342-3p, miR-197, miR-140-3p, miR-503, miR-339-3p, miR-628-3p andmiR-20a*.

According to yet another aspect the present invention provides a methodfor diagnosing non-metastatic melanoma, comprising:

-   -   (a) obtaining a sample comprising RNA from a tumor of a subject;        and    -   (b) determining, in said RNA, the presence of a plurality of        suppressive miRs comprising miRNAs selected from the group        consisting of: miR-31, miR-34a, miR-133a, miR-184, miR-185 and        miR-204,    -   wherein the presence of the plurality of said suppressive miRs        is indicative of the subject having, or being susceptible to        developing, non-metastatic melanoma.

According to one embodiment, the plurality of suppressive miRscomprising miRNAs selected from the group set forth in Table 2:

TABLE 2 Suppressive human (hsa) miRs Suppressive miRs hsa-miR-367hsa-miR-450b-5p hsa-miR-363 hsa-miR-616* hsa-miR-211 hsa-miR-500*hsa-miR-7-2* hsa-miR-148b* hsa-miR-508-3p hsa-miR-885-3p hsa-miR-507hsa-miR-551b* hsa-miR-509-5p hsa-miR-340* hsa-miR-525-5p hsa-miR-587hsa-miR-509-3-5p hsa-miR-887 hsa-miR-876-3p hsa-miR-579 hsa-miR-506hsa-miR-501-3p hsa-miR-551b hsa-miR-675 hsa-miR-184 hsa-miR-649hsa-miR-133a hsa-miR-455-5p hsa-miR-485-5p hsa-miR-487b hsa-miR-381hsa-miR-545* hsa-miR-31 hsa-miR-412 hsa-miR-146a* hsa-miR-187hsa-miR-510 hsa-miR-194 hsa-miR-23b hsa-miR-449b hsa-miR-145hsa-miR-542-5p hsa-miR-513-3p hsa-miR-192 hsa-miR-520d-5p hsa-miR-641hsa-miR-190b hsa-miR-362-3p hsa-miR-508-5p hsa-miR-513-5p hsa-miR-652hsa-miR-483-5p hsa-miR-382 hsa-miR-216a hsa-miR-582-3p hsa-miR-672hsa-miR-139-3p hsa-miR-520a-3p hsa-miR-34a hsa-miR-501-5p hsa-miR-509-3phsa-miR-204 hsa-miR-185 hsa-miR-30d hsa-miR-767-3p hsa-miR-146ahsa-miR-342-3p hsa-miR-584 hsa-miR-766 *miR generated from thecomplementary strand

In some embodiments, determining the presence of the plurality of miRNAsaccording to the methods of the invention, comprises:

(a) reverse transcribing the RNA from the sample obtained from thesubject to provide a set of target oligodeoxynucleotides;

(b) hybridizing the target oligodeoxynucleotides to a microarraycomprising oncogenic miRNA-specific probe oligonucleotides to provide ahybridization profile for the test sample; and

(c) comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample,

wherein oncogenic miRNA-specific probe oligonucleotides comprise any oneor more of the following:

-   -   (i) miRNAs selected from the group consisting of: miR-31,        miR-34a, miR-133a, miR-184, miR-185 and miR-204;    -   (ii) miRNAs selected from the group consisting of miR-374a,        miR-301a, SNORD-38b, miR-29c, miR-324-3p, miR-451, miR-29a,        miR-150, miR-342-3p, miR-197, miR-140-3p, miR-503, miR-339-3p,        miR-628-3p and miR-20a*,    -   (iii) miRNAs selected from the group set forth in Table 1; and    -   (iv) miRNAs selected from the group set forth in Table 2.    -   and wherein an increase in the signal of a plurality of        oncogenic miRNA relative to the control sample is indicative of        the subject having, or being susceptible to developing, melanoma        or aggressive melanoma. Each possibility represents a separate        embodiment of the invention.

According to yet another embodiment, the method further comprisesamplifying the target oligodeoxynucleotides prior to hybridization withthe microarray.

According to yet another aspect, the present invention provides apharmaceutical composition for treating metastatic melanoma, comprisinga plurality of suppressive miRNAs or at least one miR-agonist capable ofmimicking the activity of a plurality of suppressive miRNAs, and apharmaceutically-acceptable carrier, the plurality of suppressive miRNAscomprises miRNAs selected from the group consisting of: miR-31, miR-34a,miR-133a, miR-184, miR-185 and miR-204. Each possibility represents aseparate embodiment of the invention.

According to one embodiment, the plurality of suppressive miRNAs furthercomprises miRNAs selected from the group set forth in Table 2. Eachpossibility represents a separate embodiment of the invention.

According to another embodiment, the pharmaceutical compositioncomprises at least three suppressive miRNA and apharmaceutically-acceptable carrier.

According to yet another embodiment, the pharmaceutical compositioncomprises at least one miR-agonist capable of mimicking the activity ofthe plurality of suppressive miRNAs.

According to yet another embodiment, the at least one miR-agonist iscapable of mimicking the activity of at least three suppressive miRNAs.

According to yet another embodiment, the present invention provides amethod of treating metastatic melanoma in a subject in need thereof,comprising administering to the subject an effective amount of acomposition comprising a plurality of suppressive miRNA and apharmaceutically-acceptable carrier, the plurality of suppressive miRNAsare selected from the group consisting of miR-31, miR-34a, miR-133a,miR-184, miR-185 and miR-204. Each possibility represents a separateembodiment of the invention.

According to yet another embodiment, the plurality of suppressive miRNAsare selected from the miRNAs set forth in Table 2.

According to yet another embodiment, the method comprises administeringto the subject an effective amount of a composition comprising at leastthree suppressive miRNA. According to yet another embodiment, the methodcomprises administering to the subject an effective amount of acomposition comprising at least four suppressive miRNA.

According to yet another embodiment, the melanoma is selected from thegroup consisting of: lentigo maligna, lentigo maligna melanoma,superficial spreading melanoma, acral lentiginous melanoma, mucosalmelanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma,amelanotic melanoma and soft-tissue melanoma. Each possibilityrepresents a separate embodiment of the invention.

According to yet another embodiment, the method comprises administeringto the subject an effective amount of a composition comprising at leastone miR-agonist capable of mimicking the activity of at least threesuppressive miRNAs.

According to yet another aspect, the present invention provides apharmaceutical composition for treating metastatic melanoma, comprisingat least one compound for inhibiting expression of a plurality ofoncogenic miRNAs and a pharmaceutically-acceptable carrier, such thatproliferation of aggressive melanoma cells is inhibited, wherein theplurality of oncogenic miRNA is selected from the group set forth inTable 1.

According to one embodiment, the at least one compound is selected fromthe group consisting of double-stranded RNA, small-interfering RNA,antisense nucleic acid, antagonist of the at least one miRNA andenzymatic RNA molecules. Each possibility represents a separateembodiment of the invention.

According to another embodiment, the compound is capable of inhibitingthe expression of at least three oncogenic miRNAs.

According to yet another embodiment, at least one oncogenic miRNA ismiR-17.

According to yet another embodiment, the present invention provides amethod of treating metastatic melanoma in a subject in need thereof,comprising introducing into a cell or to a subject in need thereof aneffective amount of the at least one compound for inhibiting expressionof a plurality of oncogenic miRNAs.

According to yet another aspect the present invention provides a methodof determining responsiveness of a patient diagnosed with melanoma todrug therapy, comprising;

-   -   (a) identifying a subject diagnosed with melanoma;    -   (b) obtaining a sample comprising RNA from the subject; and    -   (c) determining, in said RNA, the presence of a plurality of        response associated miRs which are sensitive to said drug        therapy,    -   wherein the presence of at least one miR is indicative of a        positive outcome to the treatment.

According to one embodiment, drug therapy is selected from the groupconsisting of: Ipilimumab, Vemurafenib and a combination thereof Eachpossibility represents a separate embodiment of the invention.

According to yet another embodiment, the melanoma is selected from thegroup consisting of: lentigo maligna, lentigo maligna melanoma,superficial spreading melanoma, acral lentiginous melanoma, mucosalmelanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma,amelanotic melanoma and soft-tissue melanoma. Each possibilityrepresents a separate embodiment of the invention. According to yetanother embodiment the melanoma is metastatic melanoma.

According to yet another embodiment, the sample is obtained from a tumoror peripheral blood. Each possibility represents a separate embodimentof the invention.

According to yet another embodiment, responsiveness to Ipilimumab ismeasured by reduction in tumor size.

According to yet another embodiment the presence of at least one miR isdetermined by microarray analysis.

According to yet another embodiment drug therapy is combined withanother melanoma therapy including but not limited to chemotherapy,radiation therapy and surgery. Each possibility represents a separateembodiment of the invention.

These and further objects, features and advantages of the presentinvention will become apparent from the following detailed descriptionand appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the proliferation (A), invasion ability (B) andtumorigenic potential (C) of highly aggressive (HAG) and poorlyaggressive (PAG) cells.

FIG. 2 shows the relative expression ratio of miRs in HAG cells vs. PAGcells (ratio=2-ΔΔCt).

FIG. 3 exhibits differential miR expression among HAG and PAG cells,where randomly selected representative miRs falling off the diagonalline, which represent a significantly different expression level, areencircled.

FIG. 4 presents inhibition of proliferation and invasion of melanomacells by candidate suppressive miRNAs, through the fold change aboveMock-transduced cells (A), net proliferation (B), invasion ability (C)and tube formation (D) of HAG transfectants expressing suppressive miRs.

FIG. 5 demonstrates inhibition of tube formation by candidatesuppressive miRNAs in culture of Mock-transduced HAG cells (A),miRNA-transduced HAG cell (B-F). Average tube formation was quantifiedusing ImageJ analyze skeleton PlugIn (G; * denotes P<0.05, ** denotesP<0.01 (2-tailed t-test)).

FIG. 6 shows enhanced proliferation of melanoma cells by candidateoncogenic miRNA: fold enhancement by miR-17 over-expression in PAGtransductants, as compared to mock-transduced cells (A), netproliferation of the PAG transductants (B) and invasion activity of PAGtransductants (C; ** denotes P<0.01 (2-tailed t-test)).

FIG. 7 exhibits growth inhibition of melanoma xenografts obtained bytransfecting HAG transfectants expressing control (Mock; diamond) andsuppressive miR-31 (triangle), miR-34a (square), iR-184 (asterisk) andmiR-185 (circle) suppressive miRs in SCID-NOD mice, over time (A), thesize of representative tumors at day 30 (B) and the percentage of micebearing intra-abdominal macro-metastasis at day 30 (C).

FIG. 8 shows tumor growth inhibition in vivo by control (Mock; diamond)and suppressive miR-34 (circle) and miR-185 (square) miRNAs: monitoringof tumor growth in SCID-NOD mice (A, n=7; Statistical significance wastested with 2-tailed-paired t-test); Mean weight of tumors explantedupon termination of the experiment (B; Statistical significance wastested with 2-tailed t-test); Over-expression of transduced miRNAs wasconfirmed upon termination of the experiment in all tumors with qPCR.(C; * denotes P<0.05).

FIG. 9 exhibits proliferation of Mock (diamond) or mir-17(square)-transfected PAG cells.

FIG. 10 presents the expression levels, in units of ΔCt (absolute,non-relative, values) of miRs −17, −31, −34a, −133a, −184 and −185 inspecimens derived from 15 metastatic melanoma patients (A and B).

FIG. 11 shows a cluster analysis of subjects according to circulatingSNORD-38b, the miR-374 and miR-301 expression profile: (A) shows adendrogram of control subjects (C1-C4) and melanoma patients (S1-S3,S5-S8); (B) is a list of statistically significant miRs that aredifferent among controls and patients.

FIG. 12 shows a cluster analysis of subjects according to circulatingmiR-374, miR-301a and SNORD-38b represented by a Heat map of theindicated miRs in controls (C1-C10), in metastatic melanoma patients(numbers) and in a stage III patient with no evidence of disease (MA).Intense expression is denoted by darksome color, while moderateexpression is denoted by light color.

FIG. 13 is a dendrogram of control subjects (C1-C10) and melanomapatients (designated by number) for circulating miR-29c, miR-324-3p,miR-451 and miR-29a and miR-374.

FIG. 14 presents analysis of 15 miRNAs.

DETAILED DESCRIPTION OF THE INVENTION

Melanoma is a malignant tumor of melanocytes (pigment producing cells)located predominantly in skin. Although only 4% of skin cancers arediagnosed with melanoma, melanoma account to 75% of death incidencesfrom skin cancer. Unlike many other cancers, the incidence of melanomaincreased by around 160,000 per year in the Western world.

The present invention relates to methods for the diagnosis, staging andprognosis of metastatic melanoma, as well as, methods and pharmaceuticalcompositions for treating metastatic melanoma.

Melanoma according to the present invention may refer to any of thefollowing diseases: lentigo maligna, lentigo maligna melanoma,superficial spreading melanoma, acral lentiginous melanoma, mucosalmelanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma,amelanotic melanoma and soft-tissue melanoma.

The terms “malignant melanoma”, “metastatic melanoma” or “aggressivemelanoma” are interchangeable as used herein and include, but are notlimited to, superficial spreading melanoma, nodular melanoma, lentigomaligna melanoma, acral melanoma, amelanotic melanoma and desmoplasticmelanoma.

The terms “MicroRNA”, “miR” or “miRNA” are used herein interchangeablyfor describing a class of non-coding RNA molecules of 18-24 nucleotidesthat exist in a variety of organisms, including mammals, and areconserved in evolution. miRNAs can be encoded in intergenic regions,hosted within introns of pre-mRNAs or within ncRNA genes. Many miRNAsalso tend to be clustered and transcribed as polycistrons and often havesimilar spatial temporal expression patterns. MiRNAs have been found tohave roles in a variety of biological processes including developmentaltiming, differentiation, apoptosis, cell proliferation, organdevelopment, and metabolism and are major regulators of a largeproportion of animal genes or the transcriptome. The present inventionprovides patterns of microRNA molecules that specifically relate to thepresence or absence of melanoma, aggressive melanoma (oncogenic miRNAs)and indolent melanoma phenotype (suppressive miRNAs). Representatives ofthese miR patterns are shown herein to perform, in vitro and in vivo, assuppressors or inducers of aggressive melanoma.

Effective early diagnosis of melanoma, let alone, malignant melanoma ismaterial since in its early stages this disease may be cured. Thecurrent approaches for diagnosis are primarily based on pathology andimmunological staining while prognostication is mainly based on Breslowscore (depth of invasion of the primary lesion). Molecular diagnosis isadvantageous over the common methods as it can provide a reliableindication at the very early stages of onset, thereby enabling to manageand cure the disease. Further advantage of molecular diagnosis is thatit enables continuous staging of the disease, which enables to target atherapy for each stage of the disease thereby providing an improvedpatient management.

The present invention provides patterns of oncogenic microRNA moleculesthat specifically relate to the presence melanoma. As shown below, thepatterns of these microRNA of the invention strongly associate withmelanoma. Accordingly, these miRNAs are particularly suitable fordiagnosis of melanoma or for assessing susceptibility to developingmelanoma.

According to the present invention, the patterns of oncogenic miRNAswhich can be used to distinguish between healthy subjects and subjectshaving, or being susceptible to, melanoma, are circulating miRNAselected from the group consisting of: miR-150, miR-451, miR-342-3p,SNORD38b, miR-324-3p, miR-29c, miR-197, miR-140-3p, miR-29a, miR-503,miR-339-3p, miR-20a*, miR-628-3p, miR-374a and miR-301a.

The plurality of circulating miRs may be any cluster comprisingSNORD-38b with any one or more of miR-374a, miR-301, miR-29c, miR324-3p,miR-451 and miR-29a. Each possibility represents a separate embodimentof the invention.

Alternatively, the plurality of circulating miRs may be any clustercomprising miR-374a and one or more of the following circulating miRs:miR-150, miR-451, miR-342-3p, SNORD38b, miR-324-3p, miR-29c, miR-197,miR-140-3p, miR-29a, miR-503, miR-339-3p, miR-628-3p miR-20a* andmiR-301a.

In addition, the plurality of circulating miRs may be selected frommiR-374a, miR-451, miR-324-3p, miR-29c, and miR-29a.

On the other hand, miRNA which can be used to distinguish betweenpatients having metastatic melanoma and patients having non-metastaticmelanoma, are non-circulating miRNA, derived from a tumor. ThesemicroRNAs include miR-17 among the other miRNAs listed in Table 1.

Preferably, for detection of aggressive melanoma (metastatic melanoma),it is required by the principles of the present invention to detect in atumor a plurality of miRNAs selected from Table 1, wherein at least onemiRNA is miR-17.

The method of the invention for determining the presence or absence ofmelanoma in a subject may further require identifying a cluster of atleast three oncogenic miRNA, or at least four oncogenic miRNAs, from theaforementioned circulating miRNAs

Over expression of miRs which distinguishes a healthy subject from asubject having melanoma may require that the plurality of oncogenic miRsare over expressed at least 5 fold, at least 10 fold or at least 20 folddifference when comparing the two.

Preferably, the sample comprising the RNA used for determining thepresence or absence of melanoma (also termed “first sample”), based oncirculating miRNAs is obtained from peripheral blood.

In addition, the present invention provides patterns of oncogenicmicroRNA molecules that specifically relate to aggressive melanoma. Asexemplified hereinbelow, the patterns of oncogenic microRNA of theinvention strongly associate with aggressive melanoma. Moreover, theseoncogenic microRNA patterns are capable of inducing proliferation inpoorly aggressive melanoma.

Determining whether melanoma is aggressive or not may be carried outafter the person is diagnosed with melanoma according to the method ofthe invention, or before a person is diagnosed, provided that thestaging (aggressive or not) is determined according to the presence of aplurality of miRs from Table 1.

In the step of staging melanoma, the RNA is derived from a sample, alsotermed “second sample” which is typically obtained from a tumor of thesubject. The sample may contain melanocytes.

Accordingly, the oncogenic miRNAs of the invention listed in Table 1 areparticularly suitable for diagnosis of aggressive melanoma or forassessing susceptibility to developing aggressive melanoma, as well asfor molecular staging and prognostication of aggressive melanoma or fortargeting therapeutic agents to aggressive melanoma. Assessment with theoncogenic miRNAs of the invention may be accompanied by other diagnostictechniques.

The terms “susceptibility to developing aggressive melanoma” or “beingat a risk of developing melanoma and aggressive melanoma” refer to theprobability of individuals to be diagnosed with aggressive melanoma.Thus, the methods of the invention include diagnostic tools fordetecting aggressive melanoma as well as tools for predicting and/orassessing the susceptibility to develop this disease.

In one embodiment, the patterns of oncogenic miRNAs are selected fromthe group consisting of the miRNAs listed in Table 1 and combinationsthereof. Specifically, the oncogenic miRNAs are selected from the groupconsisting of the following miRNAs and combinations thereof:hsa-miR-603, hsa-miR-15a*, hsa-miR-302b*, hsa-let-7f-2*, hsa-miR-299-5p,hsa-miR-19a*, hsa-miR-7, hsa-miR-18a*, hsa-miR-886-5p, hsa-miR-183*,hsa-miR-26b*, hsa-miR-429, hsa-miR-886-3p, hsa-miR-517b, hsa-miR-142-5p,hsa-miR-942, hsa-miR-495, hsa-miR-29b-1*, hsa-miR-190, hsa-miR-572,hsa-miR-137, hsa-miR-30c-2*, hsa-miR-17*, hsa-miR-223, hsa-miR-600,hsa-miR-517a, hsa-miR-193 a-3p, hsa-miR-892b, hsa-miR-558, hsa-miR-373,hsa-miR-801, hsa-miR-92a-1*, hsa-miR-492, hsa-miR-941, hsa-miR-589,hsa-miR-616, hsa-miR-127-5p, hsa-miR-943, hsa-miR-29a*, hsa-miR-182,hsa-miR-125b-1*, hsa-miR-199b-5p, hsa-miR-200b, hsa-miR-99a*,hsa-miR-21*, hsa-miR-629, hsa-miR-539 and hsa-miR-604.

The invention also relates to methods for inhibiting or suppressingaggressive melanoma cells and tumors.

As used herein, “inhibiting or suppressing aggressive melanoma cells andtumors” refers to the inhibition or suppression of the proliferation ofa metastatic melanoma cell, namely, killing the cell, or permanently ortemporarily arresting or slowing the growth of the cell. This term alsorefers to inhibition of cell migration and invasion, thereby inhibitionor suppression of the formation of metastases. Inhibition of cellproliferation can be inferred if the number of such cells in the subjectremains constant or decreases after administration of the miR geneproducts or miR gene expression-inhibition compounds of the invention.An inhibition of cell proliferation can also be inferred if the absolutenumber of such cells increases, but the rate of tumor growth decreases.

Commonly, malignant melanoma is treated topically by surgical resection.However, this therapeutic approach is usually applicable only at earlystages, and becomes less and less effective as the disease progresses tothe metastatic stage. Atopical modalities include chemotherapy andimmunotherapy, both of which offer 10-20% response with significantadverse effects. Therapy at the molecular level, as provided by thepresent invention, confers innumerable advantages, including, targetedtherapy, tailored to the staging of the disease, thereby providing animproved patient management.

The method of the invention is based on suppression or inhibition ofaggressive melanoma by introducing into a cell or a tumor or a subject,a pharmaceutical composition comprising at least one of the following asthe pharmaceutical active ingredient: (a) a plurality, alternatively, atleast three or more, suppressive microRNA molecules; (b) agonist(s)augmenting the activity of a plurality of suppressive microRNAs; or (c)antagonist(s) inhibiting the expression of a plurality of oncogenicmiRNAs. The effective amount of the active ingredient in thepharmaceutical composition of the invention is determined duringpre-clinical trials and clinical trials by methods familiar tophysicians and clinicians. The pharmaceutical composition of theinvention further comprises a suitable carrier. The pharmaceuticalcarrier may also comprise one or more of the following: a stabilizer, asurfactant, preferably a nonionic surfactant, and optionally a saltand/or a buffering agent.

According to one embodiment, the suppressive miRNAs in thepharmaceutical composition are selected from the group consisting of themiRNAs listed in Table 2 and combinations thereof. Specifically, thesuppressive miRNAs are selected from the group consisting of thefollowing miRNAs and combinations thereof: hsa-miR-367, hsa-miR-450b-5p,hsa-miR-363, hsa-miR-616*, hsa-miR-211, hsa-miR-500*, hsa-miR-7-2*,hsa-miR-148b*, hsa-miR-508-3p, hsa-miR-885-3p, hsa-miR-507,hsa-miR-551b*, hsa-miR-509-5p, hsa-miR-340*, hsa-miR-525-5p,hsa-miR-587, hsa-miR-509-3-5p, hsa-miR-887, hsa-miR-876-3p, hsa-miR-579,hsa-miR-506, hsa-miR-501-3p, hsa-miR-551b, hsa-miR-675, hsa-miR-184,hsa-miR-649, hsa-miR-133a, hsa-miR-455-5p, hsa-miR-485-5p, hsa-miR-487b,hsa-miR-381, hsa-miR-545*, hsa-miR-31, hsa-miR-412, hsa-miR-146a*,hsa-miR-187, hsa-miR-510, hsa-miR-194, hsa-miR-23b, hsa-miR-449b,hsa-miR-145, hsa-miR-542-5p, hsa-miR-513-3p, hsa-miR-192,hsa-miR-520d-5p, hsa-miR-641, hsa-miR-190b, hsa-miR-362-3p,hsa-miR-508-5p, hsa-miR-513-5p, hsa-miR-652, hsa-miR-483-5p,hsa-miR-382, hsa-miR-216a, hsa-miR-582-3p and hsa-miR-672.

According to another embodiment, suppression or inhibition of aggressivemelanoma is achieved by introducing into a cell or a tumor or a subjectone or more agents that augment the activity of endogenous suppressivemiRs, such as, an oligonucleotide agent or small molecule agent.

Embodiments of the invention provide specific compositions and methodsthat are useful in augmenting miRNA or pre-miRNA activity levels, ine.g., a mammal, such as a human. In particular, the present inventionprovides specific compositions and methods that are useful for enhancingactivity levels of the suppressive miRNAs listed in Table 2, e.g.,miR-31, miR-34a, miR-133a, miR-184, miR-185, miR-204 and combinationsthereof.

A method of mimicking and preferably supplementing the effect of anmiRNA or pre-miRNA in a cell of a subject, using compositions(‘supermir’) that are useful in augmenting miRNA or pre-miRNA activitylevels, where the supermir is substantially single-stranded and includesa sequence that is substantially complementary to 12 to 23 contiguousnucleotides, and preferably 15 to 23 contiguous nucleotides, of a targetsequence of an miRNA or pre-miRNA nucleotide sequence is provided in USPatent Application, Publication No. 2009/031259.

The microRNA molecules of the invention can be introduced into a cell byany method known to those skilled in the art.

For example, the microRNA molecules can be injected directly into acell, such as by microinjection. Alternatively, the molecules can becontacted with a cell, preferably aided by a delivery system.

Useful delivery systems include, for example, liposomes and chargedlipids. Liposomes typically encapsulate oligonucleotide molecules withintheir aqueous center. Charged lipids generally formlipid-oligonucleotide molecule complexes as a result of opposingcharges.

These liposomes-oligonucleotide molecule complexes orlipid-oligonucleotide molecule complexes are usually internalized incells by endocytosis. The liposomes or charged lipids generally comprisehelper lipids which disrupt the endosomal membrane and release theoligonucleotide molecules.

Other methods for introducing a microRNA molecule into a cell includeuse of delivery vehicles, such as dendrimers, biodegradable polymers,polymers of amino acids, polymers of sugars, and oligonucleotide-bindingnanoparticles. In addition, pluoronic gel as a depot reservoir can beused to deliver the microRNA oligonucleotide molecules over a prolongedperiod. The above methods are described in, for example, Hughes et al.,Drug Discovery Today 6, 303-315 (2001); Liang et al. Eur. J. Biochem.269 5753-5758 (2002); and Becker et al., In Antisense Technology in theCentral Nervous System (Leslie, R. A., Hunter, A. J. & Robertson, H. A.,eds), pp. 147-157, Oxford University Press.

The suppressive microRNA molecules can be also introduced into a mammalby any method known to those in the art. An example of a suitable modeof administration includes systemic administration, enteral orparenteral. Liquid or solid (e.g., tablets, gelatin capsules)formulations can be employed. The mode of administration may includetargeting the microRNA molecule to a particular cell or tissue.Targeting of a microRNA molecule can be performed by any method known tothose skilled in the art. For example, the microRNA molecule can beconjugated to an antibody or ligand specifically recognized by receptorson the cell.

Parenteral administration of the molecules include, for exampleintravenous, intramuscular, and subcutaneous injections. A molecule maybe administered to a mammal by sustained release, as is known in theart. Sustained release administration is a method of drug delivery toachieve a certain level of the drug over a particular period of time.

Other routes of administration include intrabronchial or intranasaladministration. Intrabronchial administration can include an inhalerspray. For intranasal administration, administration of a molecule ofthe present invention can be accomplished by a nebulizer or liquid mist.

As detailed above, the molecules of the present invention can be in asuitable pharmaceutical carrier. In this specification, a pharmaceuticalcarrier is considered to be synonymous with a vehicle or an excipient asis understood by practitioners in the art. Examples of carriers includestarch, certain types of clay, gelatin, stearic acid or salts thereof,magnesium or calcium stearate, talc, vegetable fats or oils, gums andglycols.

The pharmaceutical carrier may also comprise one or more of thefollowing: a stabilizer, a surfactant, preferably a nonionic surfactant,and optionally a salt and/or a buffering agent.

According to an alternative embodiment, the method comprises introducinginto a cell or administering to the subject an effective amount of atleast one compound for inhibiting expression of the plurality ofoncogenic microRNA molecule of the invention such that proliferation ofaggressive melanoma cells is inhibited. In a particular embodiment, theat least one miR expression-inhibition compound is specific for aplurality of miRs, or at least three miRs, selected from the group setforth in Table 1.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, antagonist microRNAs, such as,antagomiRs, and enzymatic RNA molecules, such as ribozymes. Each ofthese compounds can be targeted to a given miR gene product andinterfere with the expression of (e.g., inhibit translation of, inducecleavage or destruction of) the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a short or small interfering RNA or siRNA. siRNA useful inthe present methods comprise short double-stranded RNA from about 17nucleotides to about 29 nucleotides in length, preferably from about 19to about 25 nucleotides in length. The siRNA comprise a sense RNA strandand a complementary antisense RNA strand annealed together by standardWatson-Crick base-pairing interactions (hereinafter “base-paired”). Thesense strand comprises a nucleic acid sequence that is substantiallyidentical to a nucleic acid sequence contained within the target miRgene product.

Antagomirs or “antagonist microRNA”, as used herein, refer to engineeredoligonucleotides (sometimes together with chemical modifications) thatare used to antagonize miR functions, based on complementation andhybridization.

As used herein, a nucleic acid sequence in an siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid.

As used herein, an “antisense nucleic acid” refers to a nucleic acidmolecule that binds to target RNA by means of RNA-RNA, RNA-DNA orRNA-peptide nucleic acid interactions, which alters the activity of thetarget RNA. Antisense nucleic acids suitable for use in the presentmethods are single-stranded nucleic acids (e.g. RNA, DNA, RNA-DNAchimeras, peptide nucleic acid (PNA)) that generally comprise a nucleicacid sequence complementary to a contiguous nucleic acid sequence in amiR gene product. The antisense nucleic acid can comprise a nucleic acidsequence that is 50-100% complementary, 75-100% complementary, or95-100% complementary to a contiguous nucleic acid sequence in a miRgene product. Without wishing to be bound by any theory, it is believedthat the antisense nucleic acids activate RNase H or another cellularnuclease that digests the miR gene product/antisense nucleic acidduplex.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid foruse in the present methods is a ribozyme.

Any method for determining nucleic acid sequence and for analyzing theidentified nucleotides for the presence of miRNA or an antisensethereof, known to a person skilled in the art, can be used according tothe teachings of the present invention.

This includes the method for microRNA expression analysis disclosed inU.S. Pat. No. 7,635,563. The method includes use microarrays fordetecting miRs, specifically, microRNA isolated from a sample isappended with linker(s) and a detectable labeling followed by contactinga microarray comprising at least 2 oligonucleotides with the detectablylabeled microRNA and detecting binding of the detectably labeledmicroRNA to the microarray.

In some embodiments, determining, in the presence of the plurality ofoncogenic miRNAs according to the methods of the invention, comprises:

(a) reverse transcribing the RNA from the sample obtained from thesubject, to provide a set of target oligodeoxynucleotides;

(b) hybridizing the target oligodeoxynucleotides to a microarraycomprising oncogenic miRNA-specific probe oligonucleotides, such as,miRNAs set forth in Table 1 or Table 2, to provide a hybridizationprofile for the test sample; and

(c) comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample, wherein an increase in thesignal of a plurality of oncogenic miRNA relative to the control sampleis indicative of the subject having, or being susceptible to developing,melanoma or aggressive melanoma. Each possibility represents a separateembodiment of the invention.

According to yet another embodiment, the method further comprisesamplifying the target oligodeoxynucleotides prior to hybridization withthe microarray.

The following examples are presented to provide a more completeunderstanding of the invention. The specific techniques, conditions,materials, proportions and reported data set forth to illustrate theprinciples of the invention are exemplary and should not be construed aslimiting the scope of the invention.

EXAMPLES Example 1 Identification of miRs Patterns Associated withAggressive Cancer Phenotype

The highly aggressive (HAG) C8161 and poorly aggressive (PAG) CS1-61cutaneous melanoma cell lines were derived from different metastasesfrom the same patient.

In order to identify miRNA patterns associated with aggressive phenotypeof melanoma cells, a comparative qPCR-based high throughput analysis ofhuman miRNAs (following ABI protocol) was performed on the melanomasublines C8161-HAG and C8161-PAG.

Proliferation of HAG and PAG cells was measured with standardized XTTfor 48 hours. The proliferation value for HAG cells was determined as100%. Invasion ability of HAG and PAG cells was determined by 18 hmatrigel invasion assay, which was corrected for proliferation.Tumorigenic potential of HAG and PAG cells was determined by SCinjection of 1×106 cells into SCID-NOD mice. FIG. 1 shows the mean tumorvolume within 18 days.

As shown in FIG. 1, the HAG cells are Highly Aggressive cells,manifested by high proliferation and invasion indices and by hightumorigenicity in immunodeficient mice. The PAG cells are PoorlyAggressive cells, which exert low proliferation and invasion indices andlow tumorigenic potential in immunodeficient SCID mice.

The quantity of miRNAs in HAG and PAG cells was determined by qPCR(TaqMan) and normalized to the U6 endogenous control (FIG. 2). ThesemiRNAs were expressed in both HAG and PAG cells within expression range(i.e. Ct<38; FIG. 3).

miRNAs that were not differentially expressed, or were expressed belowdetection levels in both sublines, were discarded. miRNAs that fall offthe diagonal line represent a significantly different expression level(FIG. 3). This analysis yielded a list of oncogenic and suppressivehuman miRNAs, as follows:

-   a. miRs highly expressed in HAG and low in PAG cells. These miRs    were classified as potentially “Oncogenic miRs”. A list of    suppressive miRs is provided in Table 1.-   b. miRs of low expression in HAG and high in PAG cells. These miRs    were classified as potentially “Suppressive miRs”. A list of    suppressive miRs is provided in Table 2.

Example 2 Forced Expression of Suppressive miRs Inhibit HAG Cells

Exemplar human miRs-31,-34a, -133a, -184,-185 and -204 were identifiedas suppressive miRs according to the HAG/PAG differential analysis.These miRs were cloned into PQCXIP retrovial vectors and introduced intoHAG cells. An empty vector was used as control (Mock). Transfectantswere tested in vitro in proliferation, invasion and tube formation in 3Dmatrix.

Proliferation of HAG transfectants was monitored by standardized XTT for48 h and the values of Mock-transfected HAG cells were determined as100%. Invasion ability of HAG transfectants was monitored by 18 hmatrigel invasion assay with correction for proliferation where thevalues of Mock-transfected HAG cells were determined as 100%. Tubeformation was monitored 48 h after seeding of transfectants in matrigel.

Forced expression of all of the above miRs in HAG cells resulted ininhibition of the abovementioned cancer functions (FIG. 4).

Specifically, an over-expression of at least 50-fold was conferred byreal time PCT

(FIG. 4A). The phenotype of the transduced cells was tested in vitro forproliferation, invasion and tube formation activities. Remarkably, asubstantial and consistent inhibition in net proliferation was conferredby miR-31, miR-34a, miR-184 and miR-185 as compared to the control cell(FIG. 4B) miR-204 did not inhibit the proliferation of HAG cells (FIG.4B). In contrast, miR-204 markedly inhibited the invasion activity ofHAG cells (FIG. 4C). Invasion was similarly inhibited by miR-184, butnot by the other suppressive miRNAs (FIG. 4C).

Tube formation activity was substantially inhibited by miR-34a andmiR-185, and more mildly by miR-31 and miR-184, but not by miR-204, ascompared to control (FIG. 5, A-F). Quantification of total tube lengthwas performed using ImageJ (FIG. 5G).

Importantly, the qualitative assessment of micrographic captures (FIG.5, A-F) concurred with the quantitative total length analysis (FIG. 5G).The differential effect of the miRNAs could not be simply attributed totheir differential over-expression intensities (FIG. 4A) Almost allcells were viable when assayed, as evident by <5% positive trypan bluestaining. Taken together, all live candidate suppressive miRNAs indeedexerted significant inhibitory effects on various aggressive features ofmelanoma cells. This concurs with their substantial down-regulation inthe HAG cells (FIG. 2) and their overall low expression in clinicalspecimens (FIG. 3B). This also strengthens the high throughput miRNAscreening used by the inventors and emphasizes its reliability.

Since the miRNA 17-92 clusters' functional role in cancer is wellestablished, yet never has it been tested in cutaneous melanoma, miR-17was evaluated for its effect on the aggressiveness of PAG cells. miR-17was cloned and stably over-expressed in the PAG cells. An empty vectorserved as control. A 25-fold over-expression of miR-17 was verified byreal time PCR (FIG. 6A). Importantly, miR-l7-transduced PAG cellsdisplayed a significantly enhanced proliferative activity (FIG. 6B) butnot invasive ability (FIG. 6C) or tube formation activity. These resultssupport the potentially oncogenic effects of miR-17 in melanoma.

Example 3 In vivo Expression of Suppressive miRs

miRs-31,-34a, -184 and -185 were also tested in vivo in order to confirmthe results obtained in vivo. HAG transfectants (300,000 cells/mouse)were injected SC into SCID-NOD mice. Each type of transfectant wasinjected to 6-8 mice. Formation and growth of tumor masses weremonitored 2-3 times a week using a caliper. On the day of the lastmeasurements, mice were sacrificed and tumors were extracted. Theresults indicate that tumor growth is inhibited or slowed down upontransfection with miRs-31, -34a, -184 and -185 relative to mock (FIG.7A). This observation was verified by the actual size of representativetumors extracted at day 30 of the treatment (FIG. 7B). In addition, theabdominal cavity of all mice was opened and inspected for the presenceof macro-metastases. As shown FIG. 7C the percentage of mice bearingintra-abdominal macrometastases following transfection with -34a, -184and -185 is significantly low relative to control. The numbers aboveeach bar indicate the number of mice with metastases out of the totalmice in the group (n=6-8).

The results show that forced expression of all the miRNAs of theinvention significantly inhibits HAG cell growth and spread in vivo(FIGS. 7A-C). These results further substantiate the role of thesuppressive miRNAs of the invention.

In another experiment, miRNAs were further assessed in vivo in melanomaxenograft models. The effect of the suppressive miRNAs, miR-34a andmiR-185 on tumor growth was measured following subcutaneous injection of3×10⁵ HAG transductants. Tumor masses were monitored for 28 days postinjection. Over-expression of specific miRNAs was confirmedpre-injection. Importantly, a statistically significant inhibition intumor growth was observed in both miR-34a and miR-185 transductants, ascompared to control tumors (FIG. 8A). Concurring with these results,ex-vivo weighing of tumor explants upon termination of the experimentsconfirmed that the average tumor mass of both miR-34a and miR-185transductants was lower than Mock transduced tumors (FIG. 8B). The invivo over-expression of the transduced miRNAs was confirmed in the tumorexplants (FIG. 8C). These results corroborate with the expressionresults and functional suppressive effects demonstrated in vivo (FIGS.4-5).

Example 4 Forced Expression of Oncogenic miRs Facilitate Cancer Featuresof PAG Cells

Exemplar human miR-17 was identified as an oncogenic miR according tothe HAG/PAG differential analysis. This miR was cloned into PQCXIPretrovial vectors and introduced into PAG cells. An empty vector wasused as control (Mock). Transfectants were tested in vitro forproliferation by standardized XTT assay, invasion and tube formation ina 3D matrix.

Forced expression of miR-17 in PAG cells resulted in substantialfacilitation of proliferation (FIG. 9), but not of invasion, tubeformation or in vivo tumor growth.

Example 5 Expression of Exemplar Suppressive and Oncogenic miRs inSpecimens of Metastatic Melanoma

The expression levels of miRs-17,-31,-34a, -133a, -184 and -185 weredetermined in specimens derived from 15 metastatic melanoma patients.Expression was normalized in each of the specimens to the U6 endogenouscontrol. All specimens were compared to the poorly aggressive PAG cells(FIG. 10A) or to HAG and PAG cells (FIG. 10B).

All melanoma cultures were established from distant metastases. Theanalysis established a considerable variability in miRNA expressionamong the individual specimens (FIG. 10B, black bars), mainly of miR-31(FIG. 10B). The mean expression level (FIG. 10B, horizontal lines) ofmost candidate suppressive miRNAs in the clinical specimens was betweenthe corresponding miRNA values in the PAG and HAG cells (FIG. 3B, graybars and striped bars, respectively), except for miR-185 and miR-31.While the mean level of miRNA-185 was very close to the PAG cells,miRNA-31 levels were clearly higher even than PAG cells (FIG. 10B). Incontrast, the mean expression of the candidate oncogenic miR-17 amongthe clinical specimens was even higher than in HAG cells (FIG. 10B). ThemiRNA expression patterns in clinical specimens directly shows that mostcandidate suppressive miRNAs, except for miR-31, are expressed atsignificantly lower levels than the candidate oncogenic miR-17 (FIG.10B). These results establish that the approach used to identifyfunctional suppressive and oncogenic miRNAs has physiologically-relevantgrounds.

In most cases, the expression of all tested miRs in the melanomaspecimens lies within the range between the PAG and the HAG. Thisobservation validates the expression of miRs in clinical melanomasamples. Further, this observation may be used as an inclusion criterionfor miR-based therapy (e.g. a patient with a low expression of a certainsuppressive miR is expected to benefit from induction of this miR morethan would benefit another patient having already high expression of thesame suppressive miR).

Example 6 Expression of Circulating miRs and Cluster Analysis

A comparative study including 4 healthy controls and 7 melanoma patientswas conducted. All subjects were males within the age range of 32-37y.o., without any detected co-morbidities and were not treated with anychronic medications. Total RNA was extracted from 200 ul of peripheralblood and served as template for generation of cDNA (Exiqon).Circulating miR expression was detected with LNA technology (Exiqon)using qPCR. The complete circulating miR profile was compared betweenthe two groups after normalization using an internal normalizer. Asdepicted in FIG. 11A, all healthy donors (C1-4) clustered together,while melanoma patients were clustered into two distinct groups: a) S1and S5-8 (FIG. 11A, Right); b) S2-S3 (FIG. 11A, Left), confirming thatdistinct characteristics differentiate between the healthy patients andthe melanoma patients within the miR expression data. Thirteen miRsexhibited a significantly different expression level between healthydonors and melanoma patients (FIG. 11B).

Example 7 Cluster Analysis of Subjects According to Circulating miR-374,miR-301a and SNORD-38b

A comparative expression study including 30 melanoma patients(designated 9, 23, 31, 34, 39, 41, 46-48, 51-54, 57, 62-66, 72, 74, 77,87, 90, 91, 94 and 96 in FIGS. 12) and 10 healthy individuals (controlsubjects designated C1-C10 in FIG. 12) matched for sex and age wasconducted as described above (Example 6). Three miRs displayed highdifferences that were statistically significant: miR-374, miR-301 and,SNORD-38b, which had a mean difference between the groups of 5 cycles(=64-fold), 2.8 cycles (=7-fold) and 3.9 cycles (=15-fold),respectively. Cluster analysis shows that 21 of the patients (70%) hadone of two distinct profiles (FIG. 12). In one profile, an intenseexpression was detected for the SNORD-38b, the miR-374 and the miR-301.However, in the second profile, while the SNORD-38b and the miR-374 wereexpressed intensively, the miR-301 was only moderately expressed. Noneof the healthy donors exhibited any of these two profiles (FIG. 12,light gray). A more complex combination identified 25 of the patients(83.3%), but gave a false positive of one healthy donor.

The results demonstrate that specific fingerprint patterns differentiategroups of interest, such as healthy subjects from melanoma patients,thereby facilitating the reliability of testing circulating miRs.

Example 8 Cluster Analysis of Subjects According to 15 Circulating miRs

A comparative expression study including 26 melanoma patients(designated 9, 23, 31, 34, 39, 39*, 47-48, 51-54, 57, 62-63, 65-66, 72,74, 77, 84, 85, 87, 91, 94 and 96 in FIG. 13) and 10 control subjects(designated C1-C10 in FIG. 13). It is noted that patient no. 39 and 39*refer to samples derived from the same patient but at different stagesof the disease and therefore, for the purpose of the present analysis,considered as two different samples/patients. The following miRs wereanalyzed: miR-29, miR-324p, miR-451, SNORD-38b, miR-374a, miR-150,miR-29a, miR-301a, miR-342-3p, miR-339-3p, miR-628-3p, miR-20a*,miR-503, miR-197 and miR-140-3p.

Complete statistical analysis of all subjects with respect to thefifteen miRs is shown in FIG. 14.

Five miRs displayed high differences that were statisticallysignificant: miR-29c, miR-324-3p, miR-451 and miR-29a and miR-374, asshown in Table 3:

TABLE 3 Analysis of five miRs miR T-text (control vs. patient) miR-29c0.000157446613038736 miR-324-3p 0.000518086300203002 miR-4510.0108390233049462 miR-374a 0.0492456711366369 miR-29a0.0576881855462212

A dendogram analyzing all subjects with the five significant miRs(miR-29c, miR-324-3p, miR-451, miR-374a and miR-29a) is presented inFIG. 13. As depicted, all control subjects were clustered on the toppanel of the dendogram, excluding C10, while most of the patients wereclustered in the bottom panel.

Example 9 Retrospective Comparative miR Expression Profile Analysis inMelanoma Tissue Samples of Patients Treated with Ipilimumab

Tumor tissues are obtained from patients treated with Ipilimumab, andLaser-Capture-Microdissection (LCM) is employed, thereby dissecting themelanoma tissue from melanoma tissue slides. At least 20 of the samplesare derived from patients that experienced clinical benefit fromIpilimumab, and at least 20 are derived from patients that did notrespond to the treatment. The miRs are efficiently recovered fromparaffin blocks.

Total RNA is purified from the dissected tissue samples and convertedinto cDNA. The cDNA is analyzed in 10 responders and 10 non-respondersby qPCR-based high-throughput miR analysis cards (Applied Biosystems).Expression patterns between the two groups of patients is analyzed withGenEx software and a provisional differential miR signature is selected.The provisional miR signature is further validated with specific qPCR inadditional 10 responders and 10 non-responders, thereby yielding asignature of miRs that will differentiate between Ipilimumab-respondersfrom non-responders in a statistically significant manner.

Example 10 Retrospective Comparative miR Expression Profile Analysis inMelanoma Tissue samples and Serum Samples of Patients Treated withVemurafenib

Tumor tissues are obtained from approximately 50 patients treated withVemurafenib (around 50% response). The same methodology described inExample 8 is implemented including microdissection, extraction of RNA,high throughput screening on 10 responders and 10 non-responders,analysis of the results and selection of provisional signature. Thefinal step includes validation of the provisional signature withspecific qPCR in additional 10 responders and 10 non-responders, therebyyielding a signature of miRs that differentiate betweenVemurafenib-responders from non-responders in a statisticallysignificant manner.

Peripheral blood samples are obtained from all patients at time point 0.Total RNA is purified from the serum and cDNA is generated. The entiremiR expression profile is tested in 10 responders and 10 non-responders,in order to identify a provisional miR signature in the blood. Theprovisional signature is validated in additional samples with specificqPCR, thereby yielding an independent signature of response associatedmiRs, or enhancing the accuracy of the tissue miR signature inpredicting response to treatment with Vemurafenib.

Example 11 Regulation of Target Sites by Identified miRs

The identified tissue-miRs from Examples 8 and 9 are analyzed withmiR-Path and TargetScan algorithms which predict miRs target sites. Inaddition, the identified tissue-miRs are cloned into pQCXIP vector whichare transduced into a melanoma cell line. The transduced cells aretested for proliferation, invasion through matrigel and in cytotoxicityexperiments with reactive antigen-restricted T cells. Furthermechanistic investigation is focused on the miRs that exhibit the mostpromising data, i.e. provide the strongest clinical predictive value andhave the strongest effect in vitro.

The transduced cells are tested with whole genome oligonucleotidemicroarrays and compared to melanoma cells transduced with an emptyvector. Crossing of the list of down-regulated genes with the list ofpotential targets generated by TargetScan narrows the possibilitiessignificantly. The effect on target genes is validated with qPCR. Directregulation of target sites by the miRs is verified with dual luciferase(Renilla/Firefly) assays. The effect on validated direct targets isdetermined at the protein level.

Example 12 Prospective-Retrospective Validation of the Predictive miRSignatures

Subjects are not segregated to responders/non-responders, and areblindly tested for the expression of the “relevant” signature only (3-4response associated miRs identified in Example 9 for Ipilimumab andother 3-4 response associated miRs identified in Example 10 forVemurafenib). The miR signature in the serum is tested similarly forVemurafenib only. The ability of the miR signatures to prospectivelypredict in a blind manner the response to the appropriate treatment istested prospectively and verified against the clinical outcomeretrospectively.

Example 13 Retrospective Comparative miR Expression Profile Analysis inSerum Samples Derived from Patients Treated with Ipilimumab

The entire miR expression profile is tested in 10 responders and 10non-responders, in order to identify a differential provisional miRsignature in the blood. The provisional miR signature is furthervalidated in additional samples with specific qPCR, thereby yielding anindependent signature of few response associated miRs or enhancing theaccuracy of the tissue miR signature in predicting response to treatmentwith Ipilimumab.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

1-46. (canceled)
 47. A method for diagnosing melanoma in a subject,comprising: (a) obtaining from the subject a sample of peripheral bloodcomprising RNA; and (b) determining, in said RNA, the presence of aplurality of oncogenic miRNAs, comprising miRNAs selected from the groupconsisting of: miR-374a, miR-301a, SNORD-38b, miR-29c, miR-324-3p,miR-451 and miR-29a, wherein the presence of the plurality of oncogenicmiRs is indicative of the subject having, or being susceptible todeveloping melanoma.
 48. The method of claim 47, wherein the pluralityof oncogenic miRs further comprises one or more miRs selected from thegroup consisting of: miR-150, miR-342-3p, miR-197, miR-140-3p, miR-503,miR-339-3p, miR-628-3p and miR-20a*.
 49. The method of claim 47, whereinthe plurality of oncogenic miRs is selected from the group consistingof: miR-374a, SNORD-38b, and miR-301; the plurality of oncogenic miRscomprises SNORD-38b and one or more of miR-301 and miR-374a; or theplurality of oncogenic miRs is selected from the group consisting of:miR-374a, miR-29c, miR-324-3p, miR-451 and miR-29a.
 50. The method ofclaim 48, comprising determining the presence of miR-374a and at leastone miR selected from the group consisting of miR-301a, SNORD-38b,miR-29c, miR-324-3p, miR-451, miR-29a, miR-150, miR-342-3p, miR-197,miR-140-3p, miR-503, miR-339-3p, miR-628-3p and miR-20a*.
 51. The methodof claim 48, wherein determining the presence of the plurality ofoncogenic miRNAs, comprises: (a) reverse transcribing the RNA to providea set of target oligodeoxynucleotides; (b) hybridizing the targetoligodeoxynucleotides to a microarray comprising oncogenicmiRNA-specific probe oligonucleotides selected from the group consistingof miR-374a, miR-301a, SNORD-38b, miR-29c, miR-324-3p, miR-451, miR-29a,miR-150, miR-342-3p, miR-197, miR-140-3p, miR-503, miR-339-3p,miR-628-3p and miR-20a*, to provide a hybridization profile for the testsample; and (c) comparing the test sample hybridization profile to ahybridization profile generated from a control sample, wherein anincrease in the signal of a plurality of oncogenic miRNA relative to thecontrol sample is indicative of the subject having, or being susceptibleto developing, melanoma.
 52. The method of claim 51, further comprisingamplifying the target oligodeoxynucleotides prior to hybridization withthe microarray.
 53. The method of claim 47 further comprising obtainingfrom a tumor of said subject a second sample comprising RNA anddetermining in said RNA the presence of a plurality of oncogenic miRscomprising miR-17 and at least one additional oncogenic miR, selectedfrom the group set forth in Table 1, wherein the presence of theplurality of said oncogenic miRs is indicative of the subject having, orbeing susceptible to developing, metastatic melanoma.
 54. The method ofclaim 53, comprising determining the presence of at least threeoncogenic miRNA.
 55. The method of claim 54, comprising determining thepresence of at least four oncogenic miRNAs.
 56. The method of claim 53,wherein the second sample comprises melanocytes.
 57. The method of claim47, further comprising determining, in said RNA, the presence of aplurality of suppressive miRs selected from the group set forth in Table2, wherein at least one suppressive miRNA is selected from the groupconsisting of miR-31, miR-34a, miR-133a, miR-184, miR-185 andmiR-204,wherein the presence of the plurality of said suppressive miRsis indicative of the subject having, or being susceptible to developing,non-metastatic melanoma.
 58. The method of claim 57, comprisingdetermining the presence of at least three suppressive miRNA.
 59. Themethod of claim 58, comprising determining the presence of at least foursuppressive miRNAs.
 60. The method of claim 57, wherein the samplecomprises melanocytes.
 61. A pharmaceutical composition for treatingmetastatic melanoma, comprising a plurality of suppressive miRNAs or atleast one miR-agonist capable of mimicking the activity of the pluralityof suppressive miRNAs, and a pharmaceutically-acceptable carrier,wherein the plurality of suppressive miRNAs is selected from the groupset forth in Table 2, wherein at least one suppressive miRNA is selectedfrom the group consisting of miR-31, miR-34a, miR-133a, miR-184, miR-185and miR-204.
 62. The pharmaceutical composition of claim 61, comprisingat least three suppressive miRNA and a pharmaceutically-acceptablecarrier.
 63. The pharmaceutical composition of claim 61, comprising atleast one miR-agonist capable of mimicking the activity of the pluralityof suppressive miRNAs.
 64. A method of treating metastatic melanoma in asubject in need thereof, comprising administering to the subject aneffective amount of the pharmaceutical composition of claim 61 and apharmaceutically-acceptable carrier.
 65. The method of claim 64, whereinthe melanoma is selected from the group consisting of: lentigo maligna,lentigo maligna melanoma, superficial spreading melanoma, acrallentiginous melanoma, mucosal melanoma, nodular melanoma, polypoidmelanoma, desmoplastic melanoma, amelanotic melanoma and soft-tissuemelanoma.
 66. A method of determining responsiveness of a patientdiagnosed with melanoma to drug therapy, comprising; (a) obtaining asample comprising RNA from a subject diagnosed with melanoma; and (b)determining, in said RNA, the presence of a plurality of responseassociated miRs, wherein the presence of at least one responseassociated miR is indicative of a positive outcome to the treatment.